Inflammation has emerged as a critical component of the tumor microenvironment that is required for tumor progression. Infiltrating inflammatory cells, including tumor-associated macrophages (TAMs), have been shown to promote breast cancer cell invasion and have been correlated with metastasis and poor prognosis. However, very little is known about the mechanisms regulating macrophage recruitment to the tumor microenvironment, and their role in tumor initiation due to a lack of suitable models to study preneoplastic progression. Gene profiling studies of both pre-malignant DCIS (ductal carcinoma in situ) and invasive breast cancer have resulted in the classification of various subtypes, which largely dictate patient outcome. It has been proposed that different breast cancer subtypes contain distinct cancer stem cell populations (tumor- initiating cells, TICs), which interact within a niche environment to enhance malignant progression. These TICs have been shown to be radio- and chemo-resistant, and are postulated to contribute to disease recurrence. The long-term research goal of the proposed studies is to elucidate the initiating oncogenic events that induce a pro-inflammatory environment, which is conducive for preneoplastic progression to invasive cancer. The immediate objective of this proposal is to delineate the interactions between TICs and TAMs that promote tumor initiation and progression, and how C/EBP2 mediates these interactions. The specific hypothesis tested herein is that C/EBP2 induces key inflammatory cytokines and chemokines that mediate TICs and are critical for the recruitment of TAMs during preneoplastic progression. The proposed studies will utilize two different novel preneoplastic mouse models to dissect these complex interactions. Accordingly, the following aims are proposed: Specific Aim 1: To determine whether C/EBP2-LIP is required for preneoplastic progression and tumorigenesis. Specific Aim 2: To determine whether C/EBP2-LIP is essential for TAM recruitment to preneoplastic lesions through the induction of CCL2. Specific Aim 3: To determine whether C/EBP2-LIP regulates TIC self-renewal through IL-6 signaling, and whether TAMs are required for TIC activity. The proposed studies will utilize both mouse and human inducible preneoplastic models to study the role of C/EBP2-LIP in TAM recruitment during preneoplastic progression, and to characterize the interactions between TAMs and TICs in the premalignant microenvironment. Completing these goals will enhance our understanding of the molecular mechanisms that regulate TICs in the niche microenvironment during preneoplastic progression, which will be critical for devising new treatments that selectively target therapy-resistant TICs. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Despite the recent success in the treatment of hormone-dependent breast cancers, there are currently no targeted therapies for hormone-independent breast cancers, leading to poor prognosis and decreased patient survival. A population of cells exists that is radio- and chemo-resistant, and likely contribute to disease recurrence. The results of the proposed studies will enhance our understanding of the molecular mechanisms that regulate therapy-resistant malignant cells in the niche microenvironment during preneoplastic progression, which will be critical for devising new treatments that selectively target this population.